We hypothesize that several of the factors identified in Project 1 as restriction factors of HIV replication will be components of the host antiviral sensing machinery and/or of pathways downstream this sensing machinery. Restriction factors may include known and unknown stimulators of the antiviral sensing machinery leading up to the antiviral response. There are four known families of sensing molecules of viral pathogens that induce IFN and/or antiviral responses, two of them are extracytoplasmic receptors, the Tolllike receptors (TLR) and the lectin-like receptors (LLR), and the two other are cytoplasmic receptors, the Nod-like receptors (NLR) and the RlG-1-like receptors (RLR). However, very little is known about how HIV interacts with these sensing pathways and induces and/or represses an antiviral response such as the induction of IFN and/or IFN-stimulated genes (ISGs) with anti-HIV activity. We propose to determine the restriction factors that feed into these pathways and the role they play in the anti-HIV innate immune response at the cellular level. In addition, we will study the molecular regulation of these pathways during HIV infection and their impact in the HIV antiviral response. Despite the recognized antiviral activity of type I IFN, only a few specific factors that mediate the inhibitory effect of IFN on HIV replication have been identified. While in collaboration with Projects 1, 3, 4 and 6 we will generate an HIV-1 innate immune pathway activation map that includes the restriction factors identified in this PPG, we will also select 4-5 specific restrictions factors induced by IFN to study in more detail their anti-HIV-1 activities. To achieve these goals, we have assembled a team of three co-investigators with extensive experience in these innate immunity pathways who will be responsible for accomplishing the specific aims below. Dr. Garcia-Sastre has more than 12 years of experience in IFN and the RLR pathways, and their modulation during viral infection. Dr. David has been working with the interferon system for 18 years, and his lab discovered the activation of IRF-3 by virus infection and TLR ligation as a key innate immune response pathway. Dr. Reed is a leader in the area of NLR signaling. The generated data will aide in improving understanding of the interactions of HIV with innate immune systems, and will identify novel regulators of antiviral responses that may serve as future targets for developing therapeutic strategies.